Engineered nanomedicine for myeloma and bone microenvironment targeting

Archana Swami

Michaela Reagan

Pamela Basto

Yuji Mishima

Dushanth Seevaratnam+14 authors

Supplementary Material

Supporting Information:

Click here to view.

^{Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115;}

^{Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02115;}

^{The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139;}

^{Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Harvard Medical School, Boston, MA, 02115; and}

^{King Abdulaziz University, Jeddah, Saudi Arabia}

^{To whom correspondence may be addressed. Email:}ude.dravrah.hwb.suez@dazhkorafo or ude.dravrah.icfd@lairbohg_eneri.

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved May 30, 2014 (received for review January 21, 2014)

Author contributions: A. Swami, M.R.R., O.C.F., and I.M.G. designed research; A. Swami, M.R.R., P.B., Y.M., N.K., S.G., S.Z., M. Moschetta, D.S., Y.Z., J.L., M. Memarzadeh, J.W., S.M., J.S., N.B., Z.N.L., K.N., R.B., A. Sacco, and A.M.R. performed research; A. Swami, M.R.R., R.B., A.M.R., O.C.F., and I.M.G. analyzed data; and A. Swami, M.R.R., P.B., N.K., O.C.F., and I.M.G. wrote the paper.

^{A.S. and M.R.R. contributed equally to this work.}

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved May 30, 2014 (received for review January 21, 2014)

Freely available online through the PNAS open access option.

Significance

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic breast, prostate, and blood cancers. The difficulty of eliminating bone-residing cancer necessitates novel, alternative treatments to manipulate the tumor cells and their microenvironment, with minimal off-target effects. To this end, we engineered bone-homing, stealth nanoparticles to deliver anticancer, bone-stimulatory drugs, and demonstrated their utility with bortezomib (a model drug) and multiple myeloma (a model cancer). To test our hypothesis that increasing bone volume and strength inhibits tumor growth, mice were treated with these nanoparticles before being injected with cancer cells. Results demonstrated significantly slower myeloma growth and prolonged survival. Our research demonstrates the potential of bone-homing nanomedicine as an efficacious cancer treatment mechanism.

Keywords: targeting nanomedicine, alendronate-PLGA-PEG, bone metastasis, bisphosphonate

Significance

Abstract

Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.

Abstract

The incidence of bone metastasis is common in 60–80% of cancer patients (1). During bone metastasis, cancer cells induce a sequence of changes in the microenvironment such as secreting cytokines to increase the activity of osteoclasts via the parathyroid hormone-related protein (PTHrP), receptor activator of nuclear factor-κB ligand (RANKL), and interleukin-6 (IL-6), resulting in increased bone resorption and secretion of growth factors from the bone matrix (2). This creates a “vicious cycle” of bone metastasis, where bone marrow becomes packed with cancer cells that develop resistance to conventional chemotherapy, and leads to devastating consequences of bone fractures, pain, hypercalcaemia, and spinal cord and nerve compression syndromes (2, 3). Multiple myeloma (MM) is a plasma cell cancer that proliferates primarily in bone marrow and causes osteolytic lesions (1). Antiresorption agents, such as bisphosphonates, may alleviate bone pain, but they are ineffective at inducing bone healing or osteogenesis in MM patients (4).Bortezomib is a proteasome inhibitor that has shown marked antitumor effects in patients with MM. Proteasome inhibitors, such as bortezomib, are also effective at increasing bone formation, both preclinically and clinically (5 –9). However, the major drawback of bortezomib use in early stages of MM development is its toxicity, specifically, peripheral neuropathy (5). Therefore, we aimed to develop a method to deliver bortezomib with decreased off-target side effects by using bone-specific, bortezomib-loaded nanoparticles (NPs). The NP system was based on biodegradable, biocompatible, and Food and Drug Administration (FDA)-approved components, which are both clinically and translationally relevant. NPs derived from poly(d,l-lactic-co-glycolic acid) (PLGA), a controlled release polymer system, are an excellent choice because their safety in the clinic is well established (10, 11). Polyethylene glycol (PEG)-functionalized PLGA NPs are especially desirable as PEGylated polymeric NPs have significantly reduced systemic clearance compared with similar particles without PEG (12, 13). A number of FDA-approved drugs in clinical practice use PEG for improved pharmaceutical properties such as enhanced circulation in vivo (12, 13). To target NPs to bone [rich in the mineral hydroxyapatite (HA)], the calcium ion-chelating molecules of bisphosphonates represent a promising class of ligands (14). Bisphosphonates, upon systemic administration, are found to deposit in bone tissue, preferentially at the high bone turnover sites, such as the metastatic bone lesions, with minimal nonspecific accumulation (14) and were used herein to deliver NPs to the bone.

A few systems explored for MM treatment have been tested in vitro including the following: (i) snake venom and silica NPs (15); (ii) thymoquinone and PLGA-based particles (16); (iii) curcumin and poly(oxyethylene) cholesteryl ether (PEG-Chol) NPs (17), polyethylenimine-based NPs for RNAi in MM (18), paclitaxel-Fe₃O₄ NPs (19), and liposomes (20). However, none of the above-mentioned systems have aimed to manipulate the bone marrow microenvironment rather than the myeloma cells directly (21). To date, there are no reports of using bone-targeted, controlled release, polymeric NPs with stealth properties for MM therapy. In this study, we designed NPs bearing three main components: (i) a targeting element that can selectively bind to bone mineral; (ii) a layer of stealth (PEG) to minimize immune recognition and enhance circulation; and (iii) a biodegradable polymeric material, forming an inner core, that can deliver therapeutics and/or diagnostics in a controlled manner. In this study, the physicochemical properties of a range of NPs was investigated (including NP size, charge, targeting ligand density, drug loading, and drug release kinetics) and an optimal formulation with ideal properties and maximal drug encapsulation was used for in vivo efficacy studies. We fine-tuned the NP targeting ligand density to optimize its bone-binding ability and further investigated its application for targeting myeloma in the bone microenvironment. We believe our NP system has the potential to increase drug availability by improving pharmacokinetics and biodistribution that can provide bone microenvironment specificity, which may increase the therapeutic window and most certainly decrease the off-target effects (12, 13).

Click here to view.

Acknowledgments

This work was supported by Department of Defense Grant W81XWH-05-1-0390; National Institutes of Health Grants R00 CA160350, R01 FD003743, R01 CA154648, and {"type":"entrez-nucleotide","attrs":{"text":"CA151884","term_id":"35056361"}}CA151884; Movember–Prostate Cancer Foundation Challenge Award; National Research Foundation of Korea K1A1A2048701; and the David Koch–Prostate Cancer Foundation Award in Nanotherapeutics. N.B. acknowledges Canadian Institutes of Health Research.

Acknowledgments

Footnotes

Conflict of interest statement: I.M.G. discloses her Advisory Board Membership with Novartis, Onyx, and BMS. O.C.F. discloses his financial interest in BIND Therapeutics, Selecta Biosciences, and Blend Therapeutics, three biotechnology companies developing nanoparticle technologies for medical applications. BIND, Selecta, and Blend did not support the aforementioned research, and currently these companies have no rights to any technology or intellectual property developed as part of this research.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401337111/-/DCSupplemental.

Footnotes

References

1. Roodman GDPathogenesis of myeloma bone disease. Leukemia. 2009;23(3):435–441.[PubMed][Google Scholar]
2. Reagan MR, Ghobrial IMMultiple myeloma mesenchymal stem cells: Characterization, origin, and tumor-promoting effects. Clin Cancer Res. 2012;18(2):342–349.[Google Scholar]
3. Coleman REMetastatic bone disease: Clinical features, pathophysiology and treatment strategies. Cancer Treat Rev. 2001;27(3):165–176.[PubMed][Google Scholar]
4. Garrett IR, et al Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. J Clin Invest. 2003;111(11):1771–1782.[Google Scholar]
5. Ozaki S, et al Therapy with bortezomib plus dexamethasone induces osteoblast activation in responsive patients with multiple myeloma. Int J Hematol. 2007;86(2):180–185.[PubMed][Google Scholar]
6. Giuliani N, et al The proteasome inhibitor bortezomib affects osteoblast differentiation in vitro and in vivo in multiple myeloma patients. Blood. 2007;110(1):334–338.[PubMed][Google Scholar]
7. Heider U, et al Bortezomib increases osteoblast activity in myeloma patients irrespective of response to treatment. Eur J Haematol. 2006;77(3):233–238.[PubMed][Google Scholar]
8. Zangari M, et al Response to bortezomib is associated to osteoblastic activation in patients with multiple myeloma. Br J Haematol. 2005;131(1):71–73.[PubMed][Google Scholar]
9. Terpos E, et al Increased bone mineral density in a subset of patients with relapsed multiple myeloma who received the combination of bortezomib, dexamethasone and zoledronic acid. Ann Oncol. 2010;21(7):1561–1562.[PubMed][Google Scholar]
10. Hrkach J, et al Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile. Sci Transl Med. 2012;4(128):128ra39.[PubMed][Google Scholar]
11. Kamaly N, Xiao Z, Valencia PM, Radovic-Moreno AF, Farokhzad OCTargeted polymeric therapeutic nanoparticles: Design, development and clinical translation. Chem Soc Rev. 2012;41(7):2971–3010.[Google Scholar]
12. Swami A, et al In: Multifunctional Nanoparticles for Drug Delivery Applications. Svenson S, Prud’homme RK, editors. Boston: Springer; 2012. pp. 9–29. [PubMed][Google Scholar]
13. Zhang X-Q, et al Interactions of nanomaterials and biological systems: Implications to personalized nanomedicine. Adv Drug Deliv Rev. 2012;64(13):1363–1384.[Google Scholar]
14. Zhang S, Gangal G, Uludağ H“Magic bullets” for bone diseases: Progress in rational design of bone-seeking medicinal agents. Chem Soc Rev. 2007;36(3):507–531.[PubMed][Google Scholar]
15. Sayed D, Al-Sadoon MK, Badr GSilica nanoparticles sensitize human multiple myeloma cells to snake (Walterinnesia aegyptia) venom-induced apoptosis and growth arrest. Oxid Med Cell Longev. 2012;2012:386286.[Google Scholar]
16. Ravindran J, et al Thymoquinone poly (lactide-co-glycolide) nanoparticles exhibit enhanced anti-proliferative, anti-inflammatory, and chemosensitization potential. Biochem Pharmacol. 2010;79(11):1640–1647.[Google Scholar]
17. Sou K, Oyajobi B, Goins B, Phillips WT, Tsuchida ECharacterization and cytotoxicity of self-organized assemblies of curcumin and amphiphatic poly(ethylene glycol) J Biomed Nanotechnol. 2009;5(2):202–208.[PubMed][Google Scholar]
18. Taylor CA, et al Modulation of eIF5A expression using SNS01 nanoparticles inhibits NF-κB activity and tumor growth in murine models of multiple myeloma. Mol Ther. 2012;20(7):1305–1314.[Google Scholar]
19. Yang C, et al Paclitaxel-Fe₃O₄ nanoparticles inhibit growth of CD138(−) CD34(−) tumor stem-like cells in multiple myeloma-bearing mice. Int J Nanomedicine. 2013;8:1439–1449.[Google Scholar]
20. Maillard S, et al Innovative drug delivery nanosystems improve the anti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiple myeloma. J Steroid Biochem Mol Biol. 2005;94(1-3):111–121.[PubMed][Google Scholar]
21. Cirstea D, et al Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin and perifosine induces antitumor activity in multiple myeloma. Mol Cancer Ther. 2010;9(4):963–975.[Google Scholar]
22. Azab AK, et al CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood. 2009;113(18):4341–4351.[Google Scholar]
23. Pridgen EM, et al Transepithelial transport of fc-targeted nanoparticles by the neonatal fc receptor for oral delivery. Sci Transl Med. 2013;5(213):213ra167.[Google Scholar]
24. Leleu X, et al The Akt pathway regulates survival and homing in Waldenstrom macroglobulinemia. Blood. 2007;110(13):4417–4426.[Google Scholar]
25. Dempster DW, et al Standardized nomenclature, symbols, and units for bone histomorphometry: A 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 2013;28(1):2–17.[Google Scholar]